Download University of Birmingham Mannan core branching of lipo(arabino

Mannan core branching of lipo(arabino)mannan is
required for mycobacterial virulence in the context
of innate immunity
Stoop, Esther J M; Mishra, Arun Kumar; Driessen, Nicole N; van Stempvoort, Gunny;
Bouchier, Pascale; Verboom, Theo; van Leeuwen, Lisanne M; Sparrius, Marion; Raadsen,
Susanne A; van Zon, Maaike; van der Wel, Nicole N; Besra, Gurdyal; Geurtsen, Jeroen;
Bitter, Wilbert; Appelmelk, Ben J; van der Sar, Astrid M
DOI:
10.1111/cmi.12175
License:
Creative Commons: Attribution (CC BY)
Document Version
Publisher's PDF, also known as Version of record
Citation for published version (Harvard):
Stoop, EJM, Mishra, AK, Driessen, NN, van Stempvoort, G, Bouchier, P, Verboom, T, van Leeuwen, LM,
Sparrius, M, Raadsen, SA, van Zon, M, van der Wel, NN, Besra, GS, Geurtsen, J, Bitter, W, Appelmelk, BJ &
van der Sar, AM 2013, 'Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in
the context of innate immunity' Cellular Microbiology, vol 15, no. 12, pp. 2093–2108. DOI: 10.1111/cmi.12175
Link to publication on Research at Birmingham portal
Publisher Rights Statement:
Eligibility for repository : checked 10/03/2014
General rights
Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the
copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes
permitted by law.
•Users may freely distribute the URL that is used to identify this publication.
•Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private
study or non-commercial research.
•User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?)
•Users may not further distribute the material nor use it for the purposes of commercial gain.
Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document.
When citing, please reference the published version.
Take down policy
While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been
uploaded in error or has been deemed to be commercially or otherwise sensitive.
If you believe that this is the case for this document, please contact [email protected] providing details and we will remove access to
the work immediately and investigate.
Download date: 10. Aug. 2017
Cellular Microbiology (2013) 15(12), 2093–2108
doi:10.1111/cmi.12175
First published online 22 August 2013
Mannan core branching of lipo(arabino)mannan is
required for mycobacterial virulence in the context of
innate immunity
Esther J. M. Stoop,1† Arun K. Mishra,2†
Nicole N. Driessen,1 Gunny van Stempvoort,1
Pascale Bouchier,1 Theo Verboom,1
Lisanne M. van Leeuwen,1 Marion Sparrius,1
Susanne A. Raadsen,1 Maaike van Zon,3
Nicole N. van der Wel,3 Gurdyal S. Besra,4
Jeroen Geurtsen,1 Wilbert Bitter,1,5
Ben J. Appelmelk1 and Astrid M. van der Sar1*
1
Department of Medical Microbiology and Infection
Control, VU University Medical Center, van der
Boechorststraat 7, 1081 BT Amsterdam, The
Netherlands.
2
Mycobacterial Research Division, National Institute for
Medical Research, The Ridgeway, Mill Hill, London,
NW7 1AA, UK.
3
Division of Cell Biology-B6, Netherlands Cancer
Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL),
1066 CS Amsterdam, The Netherlands.
4
School of Biosciences, University of Birmingham,
Edgbaston, Birmingham, B15 2TT, UK.
5
Department of Molecular Microbiology, VU University,
de Boelelaan 1085, 1081 HV Amsterdam, The
Netherlands.
Summary
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important
worldwide health threat. Although TB is one of the
oldest infectious diseases of man, a detailed
understanding of the mycobacterial mechanisms
underlying pathogenesis remains elusive. Here,
we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using
the Mycobacterium marinum zebrafish infection
model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in
defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)
Received 25 February, 2013; revised 3 July, 2013; accepted 17 July,
2013. *For correspondence. E-mail [email protected]; Tel.
(+31) 20 44 48296; Fax (+31) 20 44 48318.
†
These authors contributed equally to this work.
mannose branches on the lipomannan (LM) and
LAM mannan core, as determined by biochemical
analysis (NMR and GC-MS) and immunoblotting.
We found that the M. marinum mptC mutant is
strongly attenuated in embryonic zebrafish, which
rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish.
Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored
mannan core branching but not cap elongation,
was sufficient to fully complement the virulence
defect of the mptC mutant in embryos. Altogether
our data demonstrate that not LAM capping, but
mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the
context of innate immunity.
Introduction
Mycobacterium tuberculosis, the causative agent of
tuberculosis (TB), is a successful pathogen which has
infected approximately one-third of the world’s population
(Dye et al., 1999). Although a comprehensive picture
of mycobacterial factors required for pathogenesis still
remains, an essential role for the mycobacterial cell wall
has been suggested (Karakousis et al., 2004). The lipid
rich cell envelope serves as a template for glycoconjugates, such as phosphatidyl-myo-inositol mannosides
(PIMs), lipomannan (LM) and lipoarabinomannan (LAM)
(Briken et al., 2004; Mishra et al., 2011a). PIMs are composed of a mannosylphosphatidyl-myo-inositol (MPI)
anchor substituted with one to five mannose residues
(PIM1 to PIM6). Additionally, the MPI moiety can be further
covalently linked to an α(1→6)-mannopyranose (manp)
core decorated by singular α(1→2)-manp branching units
to form LM (Chatterjee et al., 1993; Mishra et al., 2011a).
LM probably serves as a precursor of LAM that is further
glycosylated by arabinan residues leading to a highly
branched arabinan domain (Birch et al., 2010; Alderwick
et al., 2011) (Fig. S1). LAM molecules can be classified
into three distinct classes, based on the structural differences of the capping motifs present on the arabinan
domain. LAM can be devoid of any capping motif, resulting in AraLAM such as in Mycobacterium chelonae
© 2013 John Wiley & Sons Ltd
cellular microbiology
2094 E. J. M. Stoop et al.
(Guerardel et al., 2002), capped by phosphoinositol (PI)
units leading to PILAM as found in Mycobacterium
smegmatis (Khoo et al., 1995), or further modified by
manp capping units resulting in ManLAM as is the case in
M. tuberculosis and Mycobacterium marinum (Appelmelk
et al., 2008; Kaur et al., 2008). Mannose caps of ManLAM
vary in length and consist of a single α(1→5)-manp that
can be elongated with one or two α(1→2)-manp units
(Besra et al., 1997; Nigou et al., 2003) (Fig. S1).
Since ManLAM is found almost exclusively in slowgrowing mycobacterial species, which include most pathogens (Nigou et al., 1997; Khoo et al., 2001; Guerardel
et al., 2003; N.N. Driessen et al., in preparation), it has
been hypothesized that mannose caps on LAM are important for mycobacterial virulence. Indeed, it has been
demonstrated that ManLAM is involved in inhibition of
phagosomal maturation (Fratti et al., 2003; Vergne et al.,
2003; Hmama et al., 2004; Kang et al., 2005) and in modulation of cytokine-mediated mechanisms of host resistance (Sibley et al., 1988; Chan et al., 1991; Knutson et al.,
1998; Nigou et al., 2001; Pathak et al., 2005). Additionally,
ManLAM is a ligand of the dendritic cell (DC)-specific
intracellular adhesion molecule-3-grabbing non-integrin
(DC-SIGN), and via this interaction it induces production of
the anti-inflammatory cytokine IL-10 (Geijtenbeek et al.,
2003; Maeda et al., 2003; Tailleux et al., 2003; Wu et al.,
2011). However, the above mentioned studies were performed with purified LAM preparations. Studies using
isogenic mycobacterial mutants defective for the α(1→5)
mannosyltransferase essential for mannose capping indicated that ManLAM does not dominate host–pathogen
interactions (Appelmelk et al., 2008; Festjens et al., 2011;
Afonso-Barroso et al., 2012), stressing the fact that such
studies need to be done using the whole bacterium instead
of purified material.
Apart from capping motifs of LAM, the mannan core of
LAM and LM has also been implicated in virulence. LM
from M. tuberculosis, which is generally longer, more
branched and has a less acylated MPI anchor than
M. smegmatis LM, inhibits host immune signalling,
whereas the M. smegmatis variant does not (Rajaram
et al., 2011; Torrelles et al., 2011). Furthermore, LM can
activate pro-inflammatory immune responses via interaction with Toll-like receptor-2 (TLR-2) (Jones et al., 2001;
Vignal et al., 2003; Dao et al., 2004; Quesniaux et al.,
2004; Nigou et al., 2008; Mishra et al., 2011b) and
potency depends on the same structural characteristics as described for interspecies variation (Nigou
et al., 2008; Rajaram et al., 2011). Interestingly, a
mutated mycobacterial LAM with a truncated arabinan
domain exhibited an increased TLR-2-dependent proinflammatory activity, which indicates that the arabinan
moiety of LAM covers the LM structure, thereby blocking
its immunostimulatory effects (Birch et al., 2010). Further-
more, using LAM and a hypermannosylated mutant LM
from Corynebacterium glutamicum, we observed that
these heterogenous molecules are recognized by
dendritic cells, mainly via TLR-2, and that mutant LM
induces stronger in vitro and in vivo cytokine responses
(Mishra et al., 2012).
The M. marinum zebrafish infection model has become
a popular model to study mycobacterial pathogenesis.
M. marinum is a close genetic relative of M. tuberculosis
(Stinear et al., 2008) and in its natural hosts, ectotherms,
it causes a TB-like disease. This disease is characterized
by the formation of granulomas; organized aggregates of
(infected) macrophages, surrounded by lymphocytes and
fibroblasts (Ramakrishnan et al., 1997; Talaat et al., 1998;
Prouty et al., 2003; van der Sar et al., 2004; Swaim et al.,
2006; Broussard and Ennis, 2007). Whereas formation
of granulomas used to be regarded as a host-driven
process, there is a growing appreciation of an active role
of the bacterium in the granuloma response. In transparent zebrafish embryos that possess only an innate
immune system, it has been shown that M. marinum promotes the formation of cellular aggregates that recapitulate host and bacterial cell properties of adult granulomas
(Davis et al., 2002; Volkman et al., 2004; Stoop et al.,
2011). Using the M. marinum zebrafish embryo model, we
recently performed a forward genetic screen to identify
mycobacterial genes involved in granuloma formation
and virulence (Stoop et al., 2011). For this, we infected
zebrafish embryos with individual M. marinum transposon
mutants and selected for mutants that were attenuated for
bacterial aggregation as compared with wildtype. In the
present work, we further characterized one of these
early granuloma mutants, which is disrupted in gene
mmar_3225. The M. tuberculosis orthologue of this
gene, Rv2181, encodes an α(1→2) mannosyltransferase
(mptC) involved in the addition of the mannose branches
on the mannan core of LM and LAM and elongation of the
mannose caps on the arabinan domain of LAM (Kaur
et al., 2008; Sena et al., 2010; Mishra et al., 2011b). We
have demonstrated that the M. marinum mptC and its
M. tuberculosis orthologue are functionally conserved and
involved in virulence in zebrafish embryos. By complementation studies we were able to separate the dual role
of the gene and established that not the length of the
mannose caps of LAM, but the presence of mannose
branching of the mannan core of LM and/or LAM is important for mycobacterial virulence in the context of innate
immunity.
Results
Identification of a M. marinum mptC mutant
We analysed 1000 mutants from a random transposon
mutant library of the fluorescently labelled M. marinum
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
2095
Fig. 1. Transposon mutant in mmar_3225 attenuated for granuloma formation in zebrafish embryo model.
A. Representative overlays of brightfield and fluorescent images of embryos at 5 dpi with wildtype M. marinum (WT), mptC mutant (mptC),
mptC mutant complemented with M. marinum mptC (mptC + mptC Mm) and mptC mutant complemented with M. smegmatis mptC
(mptC + mptC Msm). Inocula were 117, 158, 138, and 198 cfu respectively. Scale bars represent 500 μm.
B and C. Quantification of embryo infection at 5 dpi with wildtype M. marinum, the mptC mutant, the capless mutant (capless), the mptC
mutant complemented with M. marinum mptC, the mptC mutant complemented with M. smegmatis mptC and the mptC mutant complemented
with M. tuberculosis mptC (mptC + mptC Mtb). (B) Number of red pixels in fluorescent images of infected embryos as determined with
custom-designed software and (C) number of cfu of single infected embryos as determined by whole-embryo plating. Both graphs show data
of three independent experiments and the bars represent mean after log transformation. Mean ± standard deviation of inocula were 99 ± 6,
149 ± 9, 99 ± 6, 134 ± 18, 144 ± 21 and 104 ± 13 cfu respectively. *P < 0.05, ***P < 0.0001, one-way ANOVA, Bonferroni’s multiple comparison
test on log transformed data. ND, not detected.
E11 strain (Mma11) and screened for mutants with
reduced initiation of granuloma formation in embryonic
zebrafish. Upon intravenous injection of 50–200 colonyforming units (cfu) of wildtype M. marinum at 1 day post
fertilization (dpf), aggregates of intracellular bacteria are
observed at 5 days post infection (dpi) (Fig. 1A). Although
adaptive immunity is not developed at this stage, it has
been shown that these aggregates share both histological
characteristics and gene expression profiles with adult
granulomas (Davis et al., 2002; Stoop et al., 2011) and
therefore, we refer to them as early granulomas. The
screen led to the identification of 23 mutants that showed
impaired early granuloma formation (E.J.M. Stoop et al.,
in preparation). In one of the mutants, FAM223, the
transposon inserted 286 nucleotides downstream of
the start codon of gene mmar_3225, the orthologue of
the M. tuberculosis gene Rv2181 (71% identical at the
protein level). Rv2181 encodes an α(1→2) manno© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
syltransferase (MptC) required for mannose branching of
the mannan backbone of both LM and LAM and extension
of the mannose caps of LAM (Kaur et al., 2006; 2008;
Sena et al., 2010; Mishra et al., 2011b). From here on we
will refer to mutant FAM223 as mptC mutant.
Bacterial aggregate formation of the mptC mutant was
severely affected at 5 dpi (Fig. 1A). Quantification of infection was performed using two independent methods. First,
customized software was used to determine the number
of red pixels in fluorescent images of infected embryos
(Stoop et al., 2011). This analysis showed that the infection level of the mptC mutant was 17-fold reduced as
compared with wildtype M. marinum (Fig. 1B). In addition,
we determined bacterial loads per embryo by plating
infected embryos (Fig. 1C). Almost identical attenuation
levels were observed with the two methods, as the mean
bacterial load of embryos infected with the mptC mutant
was 14-fold lower than that of the wildtype. These results
2096 E. J. M. Stoop et al.
embryos. In response to infection with the complemented
mutant, bacterial aggregation and bacterial replication in
vivo were largely restored (Fig. 1A–C).
Furthermore, we analysed the ability of the M. tuberculosis mptC to restore the phenotype of the mptC mutant.
Upon expression of M. tuberculosis mptC, restoration of
the mutant phenotype was comparable with that of the
mptC mutant complemented with the M. marinum mptC,
both in terms of bacterial aggregation and replication
(Fig. 1B and C). Thus, M. marinum mptC and its M. tuberculosis orthologue are functionally conserved.
Characterization of LM and LAM of the M. marinum
mptC mutant with specific antibodies and lectins
Fig. 2. High mptC mutant inoculum restores attenuated virulence in
zebrafish embryos.
A. Representative overlays of bright field and fluorescent images of
embryos at 5 dpi with wildtype M. marinum (WT) or mptC mutant
(mptC). Scale bars represent 500 μm.
B. Number of red pixels in fluorescent images of infected embryos
as determined with custom-designed software and (C) number of
cfu of single infected embryos as determined by whole-embryo
plating. Both graphs show data of two independent experiments
and the bars represent mean after log transformation.
Mean ± standard deviation of inocula were 175 ± 81 and 419 ± 108
cfu respectively. ND, not detected.
demonstrate that MptC is involved in virulence and bacterial replication in zebrafish embryos.
To exclude that the mptC mutant is intrinsically impaired
for growth in vivo, we analysed if higher inocula could
revert the attenuated virulence phenotype to wildtype.
Indeed, we observed similar levels of infection and bacterial aggregation with the mptC mutant and the wildtype
M. marinum when 2.4-fold more mptC mutant than
wildtype bacteria were used (Fig. 2). These results indicate that the mptC mutant can replicate in vivo and that
more mptC mutant than wildtype bacteria are required for
bacterial aggregation to occur, suggesting a specific function for MptC in virulence.
Complementation of the M. marinum mptC mutant
To demonstrate that the mutant phenotype was caused
by the transposon insertion, we reintroduced the wildtype M. marinum mptC in the mptC mutant using a
mycobacterial integrative plasmid and examined the
phenotype of this complemented mutant in zebrafish
Wildtype M. marinum and the mptC mutant exhibited
identical growth characteristics on solid medium as well
as in liquid culture (data not shown). In addition, susceptibility to antibiotics (streptomycin, erythromycin, isoniazid, rifampicin, polymyxin B and chloramphenicol)
(Table S1) and protein profiles (data not shown) of the
mutant were similar as those of the parent strain,
indicating that the attenuated phenotype was not the
result of a growth defect or a gross change in cell wall
integrity.
Because it has been described that the M. tuberculosis
mptC homologue is essential for biosynthesis of the
second and possibly third manp residue of the mannose
cap of LAM, lysates of wildtype M. marinum and mptC
mutant were analysed by SDS-PAGE and immunoblot
to investigate different LAM structures. We used the
monoclonal antibodies F30-5 recognizing the arabinan
branches of LAM (α-AraLAM) (Kolk et al., 1984;
Appelmelk et al., 2008) and F55.92.1a1, indicative for
the presence of the first α(1→5)-linked manp residue on
LAM (α-ManLAM) (Appelmelk et al., 2008). Additionally,
immunoblots were probed with horseradish peroxidase
(HRP)-conjugated cyanovirin (CV-N) to detect mycobacterial di- or trimannoside capped LAM (Man2/3LAM)
(Driessen et al., 2012). As a control strain, we included
the previously described M. marinum capless mutant
which has a transposon inserted in gene mmar_2439
(capA, orthologue of M. tuberculosis Rv1635c) encoding
the α(1→5) mannosyltransferase responsible for adding
the first mannosyl residue of the mannose cap to LAM
(Dinadayala et al., 2006; Appelmelk et al., 2008).
All strains reacted well with α-AraLAM, indicating that
the basic structure of LAM is unaffected (Fig. 3A). In contrast to the capless mutant, staining with α-ManLAM was
observed for the mptC mutant, which reflects the presence of a monomannoside cap (Fig. 3B). As expected,
immunoblots probed with α-AraLAM and α-ManLAM
revealed that mptC mutant LAM migrated faster than
wildtype LAM, indicating the absence of α(1→2)-linked
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
2097
support previous reports on the role of MptC in adding
α(1→2)-manp side-chains to LM and LAM in M. tuberculosis (Kaur et al., 2008; Sena et al., 2010; Mishra
et al., 2011b). Collectively, our data indicate that the
M. marinum mptC mutant is defective in both mannose
cap extension of LAM as well as mannan core branching
of LM and LAM.
Chemical characterization of LM and LAM of the
M. marinum mptC mutant
Fig. 3. Mannose cap elongation and mannan core branching
disrupted in mptC mutant. Immunoblots of lysates of wildtype
M. marinum (WT), the mptC mutant (mptC), the capless mutant
(capl), the mptC mutant complemented with M. marinum,
M. smegmatis or M. tuberculosis mptC, (mptC + mptC Mm, Msm
and Mtb respectively). Samples were separated by SDS-PAGE and
probed with (A) Mab F30-5 (α-AraLAM), (B) Mab F55.92.1a1
(α-ManLAM), (C) CV-N-HRP or (D) DC-SIGN-Fc.
mannosyl units. Upon probing with CV-N-HRP, the
wildtype showed a clear reaction at the position of LAM,
whereas neither the mptC nor the capless mutant showed
reactivity (Fig. 3C), demonstrating the absence of the
second and third mannose capping residue. Immunoblot
analysis of the complemented mptC mutants expressing
either M. marinum or M. tuberculosis mptC resulted in
reactivity similar to that of the wildtype, both with regard to
LAM migration pattern as well as for Man2/3LAM staining
(Fig. 3A–C).
To analyse if, in addition to LAM, also LM of the mptC
mutant was affected by the mutation, we probed with a
DC-SIGN-Fc construct, because both ManLAM and LM
are DC-SIGN ligands (Maeda et al., 2003; Pitarque et al.,
2005; Torrelles et al., 2006). Immunoblots of wildtype
M. marinum probed with DC-SIGN-Fc revealed two distinct products, representing LAM and LM, as migration
patterns correspond to that of LAM and LM (Fig. 3D).
Whereas the capless mutant only lacked the upper
signal, the mptC mutant showed also no staining at the
position of LM. Introduction of either M. marinum or
M. tuberculosis mptC reverted the altered reactivity to
DC-SIGN-Fc to wildtype phenotype (Fig. 3D). These data
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
To further demonstrate the involvement of MptC in
glycoconjugate biosynthesis, we decided to analyse LAM,
LM and PIMs from the strains in detail. In accordance with
the immunoblot results, there were substantial differences
between wildtype and mptC mutant lipoglycan profiles on
SDS-PAGE (Fig. 4A). Migration patterns of the crude
extracts confirmed that LAM from the mptC mutant is
smaller in size than wildtype LAM. We observed accumulation of smaller LM in the M. marinum mptC mutant
(Fig. 4A), indicating that the LM structure is indeed
affected by the mutation similar to M. tuberculosis and
C. glutamicum (Kaur et al., 2008; Mishra et al., 2011b).
However, in one of the other studies with M. smegmatis
deletion of mptC resulted in abrogation of LM synthesis
(Kaur et al., 2006). Absence or/and presence of LM in
different species is an indication of a complicated regulation of these macromolecules in different species and
further studies are required to explain this phenomenon.
Complementation of the mptC mutant with either
M. marinum or M. tuberculosis mptC restored the wildtype
phenotype (Fig. 4B).
Crude lipoglycan preparations were further purified
using ion-exchange and gel filtration chromatography and
pure LM and LAM were analysed using nuclear magnetic
resonance (NMR). As previously described major resonance of 1H spectrum corresponding to different glycosyl
linkages are found between δ5.50 and δ4.90. Based on
previous work from ourselves (Birch et al., 2010; Mishra
et al., 2011b) and others (Nigou et al., 1999; Kaur et al.,
2008), we assigned the 13C resonance at δ101 ppm that
correlated to an anomeric proton at δ5.15 ppm to 2,6-αmanp (core) and 2-α-manp (cap). The 13C resonances at
δ105 and δ102.3, correlating to protons at δ5.07 and
δ4.90, were assigned to t-manp and 6-manp respectively.
The t-β-araf and 5-β-araf residues corresponded to
δ103.4 with 1H at δ5.16. The well-separated two spin
systems for 2-α-araf attached to the 3-position (2-αaraf→3, δ108.2, δ5.27 ppm) and the 5-position (2-αaraf→5, δ108.5, δ5.20) of the 3,5-araf were also
observed. Similarly, δ110.3 and δ5.19 ppm, δ110.3 and
δ5.14 ppm, and δ110.3 and δ5.11 ppm were assigned to
5-α-araf, 3,5-α-araf and 3,5-α-araf overlapping 5-α-araf in
different chemical environments (Nigou et al., 1999; Birch
2098 E. J. M. Stoop et al.
Fig. 4. Analysis of lipoglycan profiles confirms disrupted mannan core branching in mptC mutant.
A and B. Lipoglycans extracted from (A) wildtype M. marinum (WT) and the mptC mutant (mptC), or (B) the mptC mutant complemented with
M. marinum, M. tuberculosis or M. smegmatis mptC (mptC + mptC Mm, Mtb and Msm respectively) were run on SDS-PAGE and visualized
using Pro-Q emerald glycoprotein stain (Invitrogen). Arrows in A indicate ‘Centre of Maxima’ (CoM) of these heterogeneous lipoglycan species.
C–F. Two dimensional 1H-13C-NMR heteronuclear multiple-quantum correlation (HMQC) spectra from lipoglycans from wildtype M. marinum
and mptC mutant. LAM (C and D) and LM (E and F) were extracted and purified from M. marinum and the mptC mutant respectively, and their
2D 1H-13C-NMR HMQC spectra were recorded. Various chemical resonances were annotated to their respective linkages based on previous
work (Nigou et al., 1999; Kaur et al., 2008).
et al., 2010). In comparison to wildtype M. marinum LAM,
the mptC mutant LAM spectrum was less complex and
the resonances associated with the arabinan domain (t-βaraf, 5-β-araf, 2-α-araf→3, 2-α-araf→5, 5-α-araf, 3,5-αaraf and 3,5-α-araf) are conserved (Fig. 4C and D). As
expected, spin systems at δ101 ppm and δ5.15 ppm corresponding to 2,6-α-manp (core) and 2-α-manp (cap)
were absent in the mutant. Additionally, the spin systems
at δ105 and δ5.07 corresponding to t-manp were also
absent, while there was a concomitant increase in δ102.3
and δ4.90 assigned to 6-α-manp. Wildtype LM gave
strong signals for 2,6-α-manp and t-manp, and weak
signal for 6-α-manp. As expected, spin systems for 2,6α-manp and t-manp were almost absent with a concomitant increase in 6-α-manp in mptC mutant LM (Fig. 4E
and F). In summary, this analysis suggests that as compared with wildtype LAM, mptC mutant LAM has an unaltered arabinan domain composed of 5-α-araf branched
with 3,5-α-araf, which is further extended with 5-α-araf
leading to tetra and hexa arabinan motifs and terminated
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
Table 1. Linkage analysis of lipoglycans.
lipoglycan sample
t-αmanp
(%)
2,6-αmanp
(%)
6-αmanp
(%)
M. marinum wildtype LAM
mptC mutant LAM
mptC mutant + Mm-mptC LAM
mptC mutant + Mtb-mptC LAM
mptC mutant + Msm-mptC LAM
40
7
41
38
37
37
0
37
35
34
23
93
22
27
29
M. marinum wildtype LM
mptC mutant LM
mptC mutant + Mm-mptC LM
mptC mutant + Mtb-mptC LM
mptC mutant + Msm-mptC LM
42
8
43
40
39
38
0
38
36
36
20
92
19
24
25
Molar ratios of various mannan linkages were determined from peak
integration of GC-MS peaks. Various linkages were calculated considering total mannan content in each lipoglycan species as 100%
(Kaur et al., 2008; Birch et al., 2010). Because disruption of mptC
had no effect on arabinan linkages, only mannan core linkages are
shown here.
with t-β-araf. The mannan core of mptC mutant LAM
and LM is composed of linear 6-α-manp units without
t-manp.
Gas chromatography mass spectrometry (GC-MS) of
per-O-methylated alditol acetate derivatives confirmed
these findings and showed an unaltered glycosidic linkage profile for the arabinan domain of both wildtype
M. marinum and mptC mutant LAM and a complete loss
of branching mannan units from mptC mutant LAM and
LM (Table 1). Complementation of the mptC mutant
with either M. marinum or M. tuberculosis mptC restored
the glycosyl linkage profile to that of wildtype
(Table 1), confirming that mmar_3225 and Rv2181 are
functional orthologues. Additionally, to confirm that
the transposon insertion led to a complete inactivation of α(1→2) mannosyltransferase activity, we
performed an α(1→2) mannosyltransferase assay with
membrane fractions of the different strains and synthetic nonasaccharide acceptor, azidoethyl 6-O-benzylα- D -mannopyranosyl- (1→6)-[α- D -mannopyranosyl-(1→
6)]7-D-mannopyranoside (Acc-Man9) and the donor
polyprenyl-phosphomannose (PP-[14C]-M) as described
before (Mishra et al., 2011b). These results showed that
the disruption of mptC in M. marinum resulted in a complete abrogation of α(1→2) mannosyltransferase activity
(Fig. S2). Complementation with either M. marinum or
M. tuberculosis mptC restored this activity to wildtype
levels. Finally, we analysed PIM profiles on twodimensional thin layer chromatography (2D-TLC) and
matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and did not observe any
change in the mptC mutant as compared with wildtype M. marinum (Fig. S3 and data not shown). Collectively, our data demonstrate that both α(1→2) mannan core
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
2099
branching of LM and LAM and α(1→2) mannose cap
extension of LAM are defective in our M. marinum mptC
mutant.
Role of discrete structural LM or LAM motifs in virulence
Because the α(1→2) mannosyltransferase has a dual role
in LM and LAM biosynthesis, we attempted to define
which structural motifs of the molecules are involved in
virulence.
First, we analysed whether the absence of LAM
capping was responsible for the attenuated phenotype by
testing the M. marinum capless mutant. Previously it was
shown that the capless mutant persisted equally well as
wildtype M. marinum in macrophages and adult zebrafish
(Appelmelk et al., 2008). We examined if this was also the
case in our zebrafish embryo assay. In accordance with
the earlier reported phenotype in vitro and in adult fish,
no attenuation of the capless mutant was observed in
embryos (Fig. 1B and C). These results confirm that the
mannose caps on LAM are not essential for virulence.
We hypothesized that either the presence of a monomannoside cap on LAM or the absence of mannose
branching of the mannan core of LM and/or LAM accounts
for the attenuation of our mptC mutant.
To investigate the possibility that the attenuation was a
result of the lack of mannan backbone branching, we
complemented the mptC mutant with the M. smegmatis
mptC orthologue MSMEG_4247. Expression of this
M. smegmatis orthologue in the mptC mutant enabled
us to differentiate the two roles of M. marinum MptC. Like
its M. tuberculosis orthologue, the mannosyltransferase
encoded by MSMEG_4247 is involved in addition of
mannose branches to the LAM and LM mannan core.
However, it has no function in capping of LAM (Kaur et al.,
2006; Sena et al., 2010). Indeed, the glycosidic linkage
analysis of the mptC mutant complemented with the
M. smegmatis mptC demonstrated that mannose
branches were present on LAM (Table 1). These data
were supported by the α-ManLAM and α-AraLAM
immunoblots and migration patterns of crude extracts, as
the overall apparent molecular weight of LAM was slightly
increased as compared with that of mptC mutant LAM
(Figs 3A, B and 4B). In accordance with its predicted
function, introduction of M. smegmatis mptC did not
restore LAM capping of the mptC mutant as shown with
immunoblots probed with CV-N-HRP and DC-SIGN-Fc
(Fig. 3C and D). The DC-SIGN-Fc immunoblot, the
migration pattern of purified LM on SDS-PAGE, and
the glycosidic linkage profile also indicated that the
biosynthesis of LM in the complemented strain was comparable to that of wildtype (Figs 3D, 4B and Table 1). We
then analysed the in vivo phenotype of the mptC mutant
complemented with M. smegmatis mptC in embryos.
2100 E. J. M. Stoop et al.
Fig. 5. Initial survival of mptC mutant in macrophages not affected.
A. Overlay of differential interference contrast and fluorescent images of lateral view of the hindbrain ventricle of embryos at 4 h post infection
with wildtype M. marinum (WT) or the mptC mutant (mptC). Highly activated macrophages on the roof of the ventricle have engulfed the
bacteria. Scale bars represent 10 μm.
B. Translocation of wildtype M. marinum, the mptC mutant and the ESX-1 deficient eccCb1::tn mutant (ESX-1) in THP-1 macrophage-like
cells. Cells were infected for 24 h at MOI 10 and the percentage of cytosolic bacteria was determined by electron microscope analysis. Data
shown for wildtype and mptC mutant represent mean + SEM of two independent experiments, 465 individual bacteria in infected THP-1 were
counted. For the eccCb1::tn mutant one sample was analysed.
C. Intracellular growth of wildtype M. marinum, the mptC mutant and the eccCb1::tn mutant in THP-1 cells. Infections were performed at MOI
0.5 and colony forming units were determined by lysing infected cells and plating lysates at indicated timepoints. The graph represents
mean ± SEM of three independent replicates.
D. Intercellular spread of wildtype M. marinum, the mptC mutant and the eccCb1::tn mutant. THP-1 cells were infected at MOI 0.5 and the
percentage of infected cells was determined by microscopic enumeration of infected cells. Three representative microscopic fields with each
∼ 50–250 THP-1 cells were counted per experiment. Mean + SEM of three independent experiments is shown.
These results showed that expression of M. smegmatis
mptC restored the attenuation of the mptC mutant to
wildtype levels (Fig. 1A–C). In conclusion, our data are
consistent with the hypothesis that the attenuation of virulence of the mptC mutant is not a result of defective cap
elongation but a direct consequence of defective α(1→2)
mannosyl branching of the mannan core of LAM
and/or LM.
Analysis of the phenotype of the M. marinum mptC
mutant in macrophages
Next, we attempted to unravel the basis of attenuation
of the mptC mutant. The first step in mycobacterial
pathogenicity is the internalization of mycobacteria into
their primary host cell, the macrophage. Recruitment
of macrophages to the site of infection, as well as
phagocytosis of bacteria can be assessed in vivo by
injecting bacteria in the hind brain ventricle of embryos at
1 dpf (Herbomel et al., 2001; Davis et al., 2002). The hind
brain ventricle is a closed cavity, where macrophages are
usually absent (Herbomel et al., 2001). Ventricle infection
experiments indicated that recruitment of macrophages
to the site of infection and phagocytosis of bacteria do
not depend on MptC, as shortly after infection with both
wildtype and mptC mutant multiple macrophages were
recruited to the ventricle and internalization of bacteria
was observed (Fig. 5A).
Next, we explored the capacity of the mptC
mutant to persist inside macrophages in vitro. A strategy
used by mycobacteria to circumvent eradication in phagolysosomes is translocation from the phago(lyso)some
into the cytosol, where bacterial replication takes place
(Stamm et al., 2003; van der Wel et al., 2007). To analyse
if this step is affected in the mptC mutant, we infected
macrophages with wildtype M. marinum and the mptC
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
mutant and analysed subcellular localization of bacteria
by electron microscopy. As a control, we included the
ESX-1 deficient eccCb1::tn mutant, because a functional
ESX-1 secretion system is required for translocation (van
der Wel et al., 2007; Houben et al., 2012). The percentage of cytosolic mptC mutant bacteria was comparable
to that of the wildtype, whereas translocation of the
eccCb1::tn mutant was strongly affected (Fig. 5B and
Fig. S4).
Previous research showed that mannose-capped LAM
is required for blocking phagosomal maturation and subsequent survival of mycobacteria in macrophages (Fratti
et al., 2003; Vergne et al., 2003; Hmama et al., 2004;
Kang et al., 2005). Therefore, we infected macrophages
with wildtype M. marinum and the mptC mutant and determined intracellular replication and intercellular spread.
Again, the eccCb1::tn mutant served as a control,
because this strain is known to be attenuated for growth
and spread in macrophages (Stoop et al., 2011). Strikingly, the mptC mutant replicated and spread equally well
as the wildtype strain, while the eccCb1::tn mutant was
strongly attenuated in both assays (Fig. 5C and D).
Similar cfu counts for wildtype and mptC mutant at time 0
of infection implied that both strains are being internalized
to the same extent, which corroborates our in vivo hind
brain infection results. These experiments suggest that
the attenuated phenotype of the mptC mutant observed in
vivo is not due to defective entry or initial persistence in
macrophages.
Phenotype of the mptC mutant in adult zebrafish
The zebrafish embryo model enables us to study virulence and early granuloma formation solely in the
context of innate immunity. However, the development of
mature granulomas also depends on adaptive immunity.
To evaluate whether the mptC mutant is also attenuated
when infection is initiated in the presence of an adaptive
immune system, we performed adult zebrafish infections. Fish were infected intraperitoneally with wildtype
bacteria or the mptC mutant. Granuloma formation was
assessed by HE staining and bacterial replication was
monitored by determining colony forming units in
spleens and livers.
Infection with the mptC mutant resulted in systemic
disease with granulomas in multiple organs. Although at 4
weeks post infection (wpi) the total number of granulomas
in mptC mutant infected fish was significantly lower in
comparison to wildtype infected fish (Fig. 6A), bacterial
growth of the mptC mutant was only moderately reduced.
In spleens of fish infected with the mptC mutant the mean
bacterial load was 10-fold lower than the load of wildtype
M. marinum infected spleens, but no differences in bacterial growth in livers were observed (Fig. 6B). At 8 wpi,
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
2101
when mature granulomas had formed, comparable
numbers of granulomas were found in wildtype and mptC
mutant infected fish (Fig. 6C). The percentage of mature
granulomas, characterized by central necrosis, that are
typical for progressive disease (Swaim et al., 2006) in fish
infected with mutant bacteria was similar to that in
wildtype infected animals (44 versus 59% respectively;
P > 0.05). However, both early and late in infection, we
observed differences in the granuloma morphology in fish
infected with wildtype or mptC mutant. In mptC mutant
infected zebrafish, granulomas were generally more
compact and less tissue destruction was observed in
comparison to wildtype infected fish (Fig. 6D). Altogether,
these data demonstrate that attenuation of the mptC
mutant is less prominent in the context of an adaptive
immune system.
Discussion
Current knowledge on the role of LM and LAM in
mycobacterial virulence has mainly resulted from in vitro
studies with purified material (Nigou et al., 2001; Hmama
et al., 2004; Torrelles et al., 2011; Wu et al., 2011).
Although it has been assumed that mannose caps of LAM
are important structural features of virulent mycobacteria
to subvert the immunological defence of the host, in vivo
data indicated that mannose capping does not affect virulence (Appelmelk et al., 2008; Festjens et al., 2011;
Afonso-Barroso et al., 2012). Strikingly, Fukuda et al.
recently showed that a M. tuberculosis mptC mutant was
not attenuated for virulence in adult mice, although structural alterations in LM and LAM due to overexpression of
mptC did affect bacterial growth and virulence in vivo
(Fukuda et al., 2013). Our screen for mycobacterial
factors involved in granuloma formation led to the identification of a M. marinum mutant with an altered LM and
LAM mannosylation pattern that is strongly attenuated for
virulence in the zebrafish embryo model of infection. We
demonstrate that in this mutant, disruption of the mptC
gene results in the absence of α(1→2)-linked di- and
trimannoside caps of LAM as well as in loss of α(1→2)linked mannose branching residues on the mannan
core of LM and LAM (Fig. S1). The phenotype of the
M. marinum mptC mutant in terms of chemical structure of
LM/LAM is similar to that of a M. tuberculosis mptC
mutant (Kaur et al., 2008; Sena et al., 2010), and by complementation of the M. marinum mutant with the M. tuberculosis mptC orthologue we could demonstrate that the
two genes are functionally conserved.
The unique property of the zebrafish model to study the
first stages of granuloma formation solely in the context
of innate immunity allowed us to identify a role for LM
and/or LAM in mycobacterial virulence. Upon initiation of
infection in the context of an adaptive immune system,
2102 E. J. M. Stoop et al.
Fig. 6. MptC has a minor effect on virulence in adult zebrafish. Adult fish intraperitoneally infected with 4 × 103 cfu wildtype M. marinum (WT)
or 6 × 103 cfu mptC mutant (mptC).
A. Amount of granulomas per fish and tissue distribution at 4 wpi with wildtype M. marinum and mptC mutant. Four animals per group were
terminated and processed for histological analysis. The graph represents mean + SEM. *P < 0.05, two-way ANOVA, Bonferroni’s multiple
comparison test.
B. Bacterial load of infected fish spleens and livers at 4 wpi. Twelve fish per bacterial strain were analysed and bars indicate mean after log
transformation. ND, not detected. *P < 0.05, two-way ANOVA, Bonferroni’s multiple comparison test on log transformed data.
C. Amount of granulomas per fish and tissue distribution at 8 wpi. Two wildtype infected fish and four mptC mutant infected fish were
analysed. The graph represents mean + SEM.
D. Histopathology of fish at 8 wpi with wildtype M. marinum or mptC mutant. Representative haematoxylin and eosin (HE) staining of splenic
lesions observed at low magnification. Bars represent 100 μm.
differences between mptC mutant and wildtype
M. marinum were less apparent. Interestingly, the adult
infection data are in accordance with those found in a
study with a M. bovis BCG mptC mutant, which showed
small but significant differences in initial bacterial growth
and granuloma formation in a mice model of infection
(Festjens et al., 2011).
In order to elucidate the structural entity responsible for
the attenuation of the mptC mutant, we introduced the
M. smegmatis mptC (Kaur et al., 2006) in the mutant,
which restored mannan core branching whereas elongation of monomannoside caps was still defective (Fig. S1).
Strikingly, restoration of mannan core branching in the
mptC mutant was sufficient to fully complement the in vivo
phenotype in embryos. This demonstrates that not LAM
capping but mannan core branching of LM and/or LAM is
a virulence determining factor in the context of innate
immunity.
As an alternative explanation for the attenuated phenotype of the mptC mutant, we hypothesized that the presence of monomannoside-capped LAM is detrimental for
virulence in zebrafish embryos. Because the presence of
ManLAM in the mycobacterial cell wall has only been
described for slow-growing, pathogenic mycobacteria
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
(Nigou et al., 1997; Khoo et al., 2001; Guerardel et al.,
2003), it has been assumed that mannose caps on LAM
co-evolved with virulence. However, a recent study
revealed that in some rapid-growing non-pathogenic
mycobacterial species monomannoside caps are present
on LAM (N.N. Driessen et al., in preparation). In that
study, elongation of the cap and the presence of di- and
trimannoside caps on LAM was indeed found to be
restricted to the slow-growing species. These results may
suggest that monomannoside-capped LAM is disadvantageous for mycobacterial virulence. However, because
the in vivo phenotype of the mptC mutant complemented
with the M. smegmatis mptC did not differ considerably
from the phenotype of wildtype M. marinum or the capless
mutant, we conclude that monomannoside capped LAM
does not decrease infection capacity as compared with
fully capped or uncapped LAM. This conclusion is supported by the fact that the main capping motif of
M. marinum is monomannoside (Pitarque et al., 2005).
Yet, the data do not allow us to exclude that the attenuation of the mptC mutant is the result of a synergistic effect
of defective mannan core branching and cap elongation of LAM. It is possible that with the shortened
monomannoside capped arabinan domain, the mannan
core becomes more accessible to pattern recognition
receptors, as was previously shown for LAM of a
M. smegmatis aftC deletion mutant with a truncated
arabinan domain (Birch et al., 2010) or LAM of
M. kansasii with an enzymatically truncated arabinan
moiety (Vignal et al., 2003). However, this possibility does
not seem likely, because differences in arabinan domain
length will be negligible.
The best studied effect of purified ManLAM is its capacity to induce phagosomal maturation arrest (Fratti et al.,
2003; Vergne et al., 2003; Hmama et al., 2004; Kang
et al., 2005). We analysed entry, subcellular localization,
replication and spread of the mptC mutant in
macrophages. Surprisingly, all macrophage experiments
showed that the mptC mutant did not differ from the
wildtype strain. Because a reduced ability to arrest
phagosomal maturation would probably affect intracellular
survival, we conclude that phagosomal maturation arrest
of the mptC mutant is not significantly affected. Our data
are in agreement with a study demonstrating that the
M. bovis BCG mptC mutant did not differ from wildtype
M. bovis BCG in vitro (Festjens et al., 2011).
In addition to effects on phagosomal maturation,
ManLAM has been shown to bind DC-SIGN, thereby
suppressing DC maturation and skewing cell mediated
immunity to interfere with induction of a protective
anti-TB immune response (Geijtenbeek et al., 2003;
Maeda et al., 2003; Tailleux et al., 2003; Wu et al.,
2011). We show that binding of a DC-SIGN-Fc construct
to both LAM and LM in lysates of the mptC mutant
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
2103
was affected. This confirms that the DC-SIGN-LAM
binding depends on mannose capping residues and
indicates that mannose branches on LM are important
for DC-SIGN-LM interaction. However, it remains to
be determined whether binding of intact bacteria to
DC-SIGN is impaired, as for unknown reasons, binding
of intact M. marinum bacteria to DC-SIGN expressed on
human DCs cannot be detected (Pitarque et al., 2005;
Appelmelk et al., 2008). Moreover, because attenuation
of the mptC mutant is primarily observed in the context
of innate immunity, and zebrafish DCs are not detected
before 5–7 dpf (Lugo-Villarino et al., 2010; Wittamer
et al., 2011), it is unlikely that the observed phenotype
results from a disrupted DC-SIGN binding and/or DC
modulation.
It seems reasonable to assign the mutant phenotype
to defective mannan core α(1→2) branching. Multiple
studies have emphasized the potency of the mannan
core of both LM and (truncated) LAM to induce innate
TLR-2 responses (Vignal et al., 2003; Dao et al., 2004;
Quesniaux et al., 2004; Doz et al., 2007; Nigou et al.,
2008; Birch et al., 2010). The level of mannan core
branching is one of the structural features that determines TLR-2 activity (Nigou et al., 2008). Further studies
are required to determine if attenuation of the mptC
mutant can be attributed to enhanced TLR-2 signalling.
Unfortunately, as a single mannosyltransferase is
responsible for both branching of the mannan core and
elongation of the mannose cap, it is not possible to
separate these functions and study the effect of a
mutant exclusively disrupted in mannan core branching.
It is also impossible to identify whether mutated LM or
LAM is the major contributor to the observed in vivo
phenotype.
Despite the role of LM/LAM during early granuloma formation, our data do not strengthen the idea of
utilization of such a mutant strain for the development
of novel vaccines, especially if this mutation is not
combined with other mutations. It is intriguing though
that in the M. marinum zebrafish infection model an
effect of LM and/or LAM mannan core branching on
virulence can be shown. Our study demonstrates for the
first time that mannan core branching of LM and/or LAM
is important for virulence in the context of innate immunity and confirms that the immunological properties
assigned to purified mannose capped LAM are irrelevant
in vivo.
Experimental procedures
Bacteria and growth conditions
Mycobacterium marinum strain E11 and the Escherichia coli
strain DH5α were cultured as previously described (Stoop et al.,
2011).
2104 E. J. M. Stoop et al.
Identification of genes involved in early granuloma
formation in zebrafish embryos
A transposon mutant library was generated in M. marinum strain
E11 (Mma11) containing pSMT3-mcherry and 1000 individual
mutants were screened for their ability to initiate granuloma formation in embryonic zebrafish (Danio rerio) using our previously
described method (Stoop et al., 2011). Briefly, embryos were
infected at ∼ 28 h post fertilization with 50–200 colony forming
units of bacterial suspension injected into the caudal vein. At 5
dpi infected embryos were monitored with fluorescence microscopy. Bacterial mutants that repeatedly showed significantly
reduced early granuloma formation as compared with the
wildtype M. marinum, as determined by custom-designed software, were selected. Transposon insertion sites were determined
by ligation-mediated PCR and sequence analysis. All procedures
involving zebrafish embryos were performed in compliance with
local animal welfare laws.
Determination of bacterial loads of infected embryos
Embryo lysates were prepared as described previously (Stoop
et al., 2011). Shortly, embryos were dissociated in the presence
of 5% (w/v) SDS, lysed and decontaminated by incubation with
MycoPrep reagent (BD Bioscience), neutralized with phosphate
buffered saline (PBS) and serial dilutions were plated.
Immunoblotting assay
Bacteria grown to exponential phase were lysed by bead-beating
(Biospec). Samples were normalized to total protein concentration using BCA Protein Assay (Pierce), separated by SDS-PAGE,
transferred to a PVDF membrane (Millipore) and immunostained
as previously described (Appelmelk et al., 2008; Driessen et al.,
2010; 2012).
Chemical analysis of LM and LAM
Polar and apolar lipids were extracted and examined by two
dimensional thin-layer chromatography (2D-TLC) on aluminum
backed plates of silica gel 60 F254 (Merck 5554), using CHCl3/
CH3OH/H2O (65:25:4, v/v/v) in the first direction and CHCl3/
CH3COOH/CH3OH/H2O (40:25:3:6, v/v/v/v) in the second
direction as previously described (Dobson et al., 1995).
Glycolipids were visualized by spraying plates with α-naphthol/
sulfuric acid, followed by gentle charring of the plates.
Lipoglycans were extracted from delipidated cells as previously
described (Nigou et al., 1997; Ludwiczak et al., 2001; 2002) and
crude lipoglycan extracts were further subjected to Octyl
Sepharose CL-4B and Sephacryl S-200 (2.5 cm × 50 cm) gel
filtration chromatography to separate LM and LAM in different
fractions (Mishra et al., 2011b). For determining the sugar linkages, lipoglycans were per-O-methylated, hydrolysed, reduced
and per-O-acetylated. The resulting alditol acetates were analysed and identified using GC-MS as described previously
(Tatituri et al., 2007). NMR spectra were recorded on a Bruker
DMX-500 equipped with a double resonance (1H/X)-BBi
z-gradient probe head. All samples were exchanged in D2O (D,
99.97% from Euriso-top, Saint-Aubin, France), with intermediate
lyophilization, and then dissolved in 0.5 ml D2O and analysed at
313 K. The 1H and 13C NMR chemical shifts were referenced
relative to internal acetone at 2.225 and 34.00 ppm respectively.
All the details concerning NMR sequences used and experimental procedures were described in previous studies (Gilleron et al.,
2000).
In vitro α(1→2)-mannopyranosyltransferase assay
α(1→2)-Mannosyltransferase activity was determined using
synthetic nonasaccharide acceptor, azidoethyl 6-O-benzylα- D -mannopyranosyl-(1→6)-[α- D -mannopyranosyl-(1→6)]7 - D mannopyranoside (Acc-Man9) and the donor polyprenylphosphomannose (PP-[14C]-M) in a cell-free assay using 1 mg of
membrane protein as described previously (Mishra et al., 2011b).
The products of the assay were resuspended in water before
scintillation counting. The incorporation of [14C]-Manp was
determined by subtracting counts present in control assays.
Blank = synthetic nonasaccharide acceptor, Acc-Man9 and the
donor polyprenyl-phosphomannose (PP-[14C]-M without any
membrane.
Complementation of the mptC mutant
The wildtype mmar_3225 gene was PCR amplified with
M. marinum Mma11 genomic DNA as a template using primers
mmar_3225-F: aGATATCaggtgcgggtcacaaaaa and mmar_
3225-R: aGATATCatcaagaacggcggcaag (uppercase, engineered
EcoRV sites). The PCR product was digested with EcoRV and
ligated into the StuI site of the integrative and chloramphenicol
resistant vector pUC-INT-CAT (Abdallah et al., 2009). The
M. tuberculosis and M. smegmatis orthologues Rv2181 and
MSMEG_4247 were amplified from the M. tuberculosis H37Rv
or M. smegmatis mc2155 genome, respectively, using primers
Rv2181-F: TTTAAAgacggtggataaaccgtacaa and Rv2181-R:
TTTAAAggtgcaaatcccaattcaac or MSMEG4247-F: GATATCat
cacgacgtggtggatgt and MSMEG4247-R: TTTAAAacgtgaagaag
gcaggtgac (uppercase, DraI and EcoRV sites). PCR products
were ligated into the pJET1.2 cloning vector (Fermentas),
excised with DraI alone or in combination with EcoRV, and the
resulting blunt-ended inserts were ligated into pUC-INT-CAT
digested with StuI. Resulting plasmids were introduced into the
mptC mutant by electroporation as described previously (Stoop
et al., 2011).
Hind brain ventricle infection
Wildtype zebrafish embryos and bacteria were treated as
described for caudal vein infection (Stoop et al., 2011). At ∼ 28 h
post fertilization embryos were removed from their chorion and
infected by microinjection in the hind brain ventricle. At 4 h post
infection embryos were monitored by a ZEISS Axiovert 200 Marianas™ digital imaging microscope as described previously (van
der Sar et al., 2003).
Infection of THP-1 cells and electron microscopy
Infection of THP-1 monocytic cells was executed as previously
described (Stoop et al., 2011). Briefly, for analysis of intracellular
growth and intercellular spread, THP-1 cells were differentiated
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
into macrophage-like cells with phorbol myristate acetate (Sigma)
and seeded at a density of 3 × 105 cells ml−1 in 24-well plates in
the absence or presence of glass coverslips respectively. Bacterial suspensions were passed through a 5 μm filter to remove
clumps, added to the cells at a multiplicity of infection (MOI) of 0.5
and incubated for 2 h at 30°C and 5% CO2. Extracellular bacteria
were removed by washing with RPMI medium and cells were
incubated with fresh medium for the indicated time points at
30°C. For determination of intracellular growth, cells were lysed
with 1% Triton X-100 and serial dilutions were plated. To analyse
intercellular spread, infected cells on coverslips were fixed with
paraformaldehyde and examined microscopically. Samples for
electron microscopy were prepared as described previously (van
der Wel et al., 2007). Differentiated THP-1 cells were infected at
a MOI of 10 in 75 cm2 cell culture flasks (Corning), extracellular
bacteria were removed by washing infected cells with medium
and at 24 h post infection cells were fixed for 2 h with paraformaldehyde and glutaradehyde in PHEM buffer. Cell sections of
60 nm thick, prepared at −120°C, were immunogold-labelled with
protein A conjugated to 10 nm gold and primary antibody against
LAMP-1 (clone H4A3 from BD Biosciences) or CD63 (M1544
from Sanquin). Sections were examined with a FEI CM10 transmission electron microscope and translocation was quantified in
a double blinded fashion.
Adult zebrafish experiment
Adult zebrafish infections were performed following procedures
described previously (van der Sar et al., 2004) and approved by
the local Animal Welfare Committee, under protocol number
MM01-02 and MM10-01. Bacteria were declumped with 0.3%
Tween-80 in PBS and resuspended in PBS. Wildtype male
zebrafish of ∼ 1 year old were anaesthetized in 0.02% (w/v)
ethyl-3-aminobenzoate methanesulfonate salt (Sigma) and
injected intraperitoneally with 10 μl of bacterial suspension.
Control fish were injected with the same volume of PBS. Fish
were sacrificed by incubation in a high concentration (0.05%) of
the anaesthetic. Bacterial counts in livers and spleens of 12 fish
per group were evaluated by plating serial dilutions of homogenized organ samples decontaminated with MycoPrep reagent
(BD Bioscience) onto 7H10 agar plates. For histopathological
examination, fish were terminated and preserved in Dietrich’s
fixative (30% ethanol, 10% formalin, 2% glacial acetic acid in
deionized water) for > 48 h before embedding in paraffin and
sectioning. Frontal sections (4–7 μm) were stained with
haematoxylin and eosin and observed for signs of infection and
granuloma formation with a Zeiss Axioskop light microscope.
Images were generated with a Leica DC500 camera.
Acknowledgements
This work was supported by the Smart Mix Program of the
Netherlands Ministry of Economic Affairs and the Netherlands
Ministry of Education, Culture and Science. GSB acknowledges
support in the form of a Personal Research Chair from Mr James
Bardrick, Royal Society Wolfson Research Merit Award, as a
former Lister Institute-Jenner Research Fellow, the Medical
Research Council and the Wellcome Trust (081569/Z/06/Z).
JG is financially supported by the Netherlands Organization
for Scientific Research (NOW) through a VENI research
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
2105
grant (016.101.001). We thank Sylvia Bogaards, Eveline
Weerdenburg, Nina Hertoghs, Robert van de Weerd for technical
support, Wim Schouten for his expert technical assistance and
Birgit Witte for help with statistical analyses.
Conflict of interest
The authors declare no conflicts of interest of any kind.
References
Abdallah, A.M., Verboom, T., Weerdenburg, E.M.,
Gey van Pittius, N.C., Mahasha, P.W., Jimenez, C., et al.
(2009) PPE and PE_PGRS proteins of Mycobacterium
marinum are transported via the type VII secretion system
ESX-5. Mol Microbiol 73: 329–340.
Afonso-Barroso, A., Clark, S.O., Williams, A., Rosa,
G.T., Nobrega, C., Silva-Gomes, S., et al. (2012)
Lipoarabinomannan mannose caps do not affect
mycobacterial virulence or the induction of protective
immunity in experimental animal models of infection and
have minimal impact on in vitro inflammatory responses.
Cell Microbiol 15: 660–674.
Alderwick, L.J., Lloyd, G.S., Ghadbane, H., May, J.W., Bhatt,
A., Eggeling, L., et al. (2011) The C-terminal domain of the
Arabinosyltransferase Mycobacterium tuberculosis EmbC
is a lectin-like carbohydrate binding module. PLoS Pathog
7: e1001299.
Appelmelk, B.J., den Dunnen, J., Driessen, N.N., Ummels,
R., Pak, M., Nigou, J., et al. (2008) The mannose cap of
mycobacterial lipoarabinomannan does not dominate the
Mycobacterium-host interaction. Cell Microbiol 10: 930–
944.
Besra, G.S., Morehouse, C.B., Rittner, C.M., Waechter, C.J.,
and Brennan, P.J. (1997) Biosynthesis of mycobacterial
lipoarabinomannan. J Biol Chem 272: 18460–18466.
Birch, H.L., Alderwick, L.J., Appelmelk, B.J., Maaskant, J.,
Bhatt, A., Singh, A., et al. (2010) A truncated lipoglycan
from mycobacteria with altered immunological properties.
Proc Natl Acad Sci USA 107: 2634–2639.
Briken, V., Porcelli, S.A., Besra, G.S., and Kremer, L. (2004)
Mycobacterial lipoarabinomannan and related lipoglycans:
from biogenesis to modulation of the immune response.
Mol Microbiol 53: 391–403.
Broussard, G.W., and Ennis, D.G. (2007) Mycobacterium
marinum produces long-term chronic infections in medaka:
a new animal model for studying human tuberculosis.
Comp Biochem Physiol C Toxicol Pharmacol 145: 45–54.
Chan, J., Fan, X.D., Hunter, S.W., Brennan, P.J., and
Bloom, B.R. (1991) Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium
tuberculosis within macrophages. Infect Immun 59: 1755–
1761.
Chatterjee, D., Khoo, K.H., McNeil, M.R., Dell, A., Morris,
H.R., and Brennan, P.J. (1993) Structural definition of the
non-reducing termini of mannose-capped LAM from Mycobacterium tuberculosis through selective enzymatic degradation and fast atom bombardment-mass spectrometry.
Glycobiology 3: 497–506.
Dao, D.N., Kremer, L., Guerardel, Y., Molano, A.,
Jacobs, W.R., Jr, Porcelli, S.A., and Briken, V. (2004)
2106 E. J. M. Stoop et al.
Mycobacterium tuberculosis lipomannan induces apoptosis and interleukin-12 production in macrophages. Infect
Immun 72: 2067–2074.
Davis, J.M., Clay, H., Lewis, J.L., Ghori, N., Herbomel, P.,
and Ramakrishnan, L. (2002) Real-time visualization of
mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity 17: 693–702.
Dinadayala, P., Kaur, D., Berg, S., Amin, A.G., Vissa, V.D.,
Chatterjee, D., et al. (2006) Genetic basis for the synthesis
of the immunomodulatory mannose caps of lipoarabinomannan in Mycobacterium tuberculosis. J Biol Chem 281:
20027–20035.
Dobson, G., Christie, W.W., and Nikolova-Damyanova, B.
(1995) Silver ion chromatography of lipids and fatty acids.
J Chromatogr B Biomed Appl 671: 197–222.
Doz, E., Rose, S., Nigou, J., Gilleron, M., Puzo, G., Erard, F.,
et al. (2007) Acylation determines the toll-like receptor
(TLR)-dependent positive versus TLR2-, mannose
receptor-, and SIGNR1-independent negative regulation of
pro-inflammatory cytokines by mycobacterial lipomannan.
J Biol Chem 282: 26014–26025.
Driessen, N.N., Stoop, E.J., Ummels, R., Gurcha, S.S.,
Mishra, A.K., Larrouy-Maumus, G., et al. (2010) Mycobacterium marinum MMAR_2380, a predicted transmembrane
acyltransferase, is essential for the presence of the
mannose cap on lipoarabinomannan. Microbiology 156:
3492–3502.
Driessen, N.N., Boshoff, H.I., Maaskant, J.J., Gilissen, S.A.,
Vink, S., van der Sar, A.M., et al. (2012) Cyanovirin-N
inhibits mannose-dependent mycobacterium-C-type lectin
interactions but does not protect against murine tuberculosis. J Immunol 189: 3585–3592.
Dye, C., Scheele, S., Dolin, P., Pathania, V., and Raviglione,
M.C. (1999) Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by
country. WHO Global Surveillance and Monitoring Project.
JAMA 282: 677–686.
Festjens, N., Bogaert, P., Batni, A., Houthuys, E., Plets, E.,
Vanderschaeghe, D., et al. (2011) Disruption of the SapM
locus in Mycobacterium bovis BCG improves its protective
efficacy as a vaccine against M. tuberculosis. EMBO Mol
Med 3: 222–234.
Fratti, R.A., Chua, J., Vergne, I., and Deretic, V. (2003) Mycobacterium tuberculosis glycosylated phosphatidylinositol
causes phagosome maturation arrest. Proc Natl Acad Sci
USA 100: 5437–5442.
Fukuda, T., Matsumura, T., Ato, M., Hamasaki, M., Nishiuchi,
Y., Murakami, Y., et al. (2013) Critical roles for lipomannan and lipoarabinomannan in cell wall integrity of
mycobacteria and pathogenesis of tuberculosis. MBio 4:
e00472-00412.
Geijtenbeek, T.B., Van Vliet, S.J., Koppel, E.A.,
Sanchez-Hernandez, M., Vandenbroucke-Grauls, C.M.,
Appelmelk, B., and Van Kooyk, Y. (2003) Mycobacteria
target DC-SIGN to suppress dendritic cell function. J Exp
Med 197: 7–17.
Gilleron, M., Bala, L., Brando, T., Vercellone, A., and Puzo, G.
(2000) Mycobacterium tuberculosis H37Rv parietal and
cellular lipoarabinomannans. Characterization of the acyland glyco-forms. J Biol Chem 275: 677–684.
Guerardel, Y., Maes, E., Elass, E., Leroy, Y., Timmerman, P.,
Besra, G.S., et al. (2002) Structural study of lipomannan
and lipoarabinomannan from Mycobacterium chelonae.
Presence of unusual components with alpha 1,3mannopyranose side chains. J Biol Chem 277: 30635–
30648.
Guerardel, Y., Maes, E., Briken, V., Chirat, F., Leroy, Y., Locht,
C., et al. (2003) Lipomannan and lipoarabinomannan from
a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties. J Biol
Chem 278: 36637–36651.
Herbomel, P., Thisse, B., and Thisse, C. (2001) Zebrafish
early macrophages colonize cephalic mesenchyme and
developing brain, retina, and epidermis through a M-CSF
receptor-dependent invasive process. Dev Biol 238: 274–
288.
Hmama, Z., Sendide, K., Talal, A., Garcia, R., Dobos, K.,
and Reiner, N.E. (2004) Quantitative analysis of
phagolysosome fusion in intact cells: inhibition by
mycobacterial lipoarabinomannan and rescue by an
1alpha,25-dihydroxyvitamin D3-phosphoinositide 3-kinase
pathway. J Cell Sci 117: 2131–2140.
Houben, D., Demangel, C., van Ingen, J., Perez, J., Baldeon,
L., Abdallah, A.M., et al. (2012) ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria.
Cell Microbiol 14: 1287–1298.
Jones, B.W., Means, T.K., Heldwein, K.A., Keen, M.A., Hill,
P.J., Belisle, J.T., and Fenton, M.J. (2001) Different Toll-like
receptor agonists induce distinct macrophage responses.
J Leukoc Biol 69: 1036–1044.
Kang, P.B., Azad, A.K., Torrelles, J.B., Kaufman, T.M.,
Beharka, A., Tibesar, E., et al. (2005) The human
macrophage mannose receptor directs Mycobacterium
tuberculosis lipoarabinomannan-mediated phagosome
biogenesis. J Exp Med 202: 987–999.
Karakousis, P.C., Bishai, W.R., and Dorman, S.E. (2004)
Mycobacterium tuberculosis cell envelope lipids and the
host immune response. Cell Microbiol 6: 105–116.
Kaur, D., Berg, S., Dinadayala, P., Gicquel, B., Chatterjee, D.,
McNeil, M.R., et al. (2006) Biosynthesis of mycobacterial
lipoarabinomannan: role of a branching mannosyltransferase. Proc Natl Acad Sci USA 103: 13664–
13669.
Kaur, D., Obregon-Henao, A., Pham, H., Chatterjee, D.,
Brennan, P.J., and Jackson, M. (2008) Lipoarabinomannan
of Mycobacterium: mannose capping by a multifunctional
terminal mannosyltransferase. Proc Natl Acad Sci USA
105: 17973–17977.
Khoo, K.H., Dell, A., Morris, H.R., Brennan, P.J., and
Chatterjee, D. (1995) Inositol phosphate capping of the
nonreducing termini of lipoarabinomannan from rapidly
growing strains of Mycobacterium. J Biol Chem 270:
12380–12389.
Khoo, K.H., Tang, J.B., and Chatterjee, D. (2001) Variation
in mannose-capped terminal arabinan motifs of
lipoarabinomannans from clinical isolates of Mycobacterium tuberculosis and Mycobacterium avium complex.
J Biol Chem 276: 3863–3871.
Knutson, K.L., Hmama, Z., Herrera-Velit, P., Rochford, R.,
and Reiner, N.E. (1998) Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
Mannosyltransferase affects granuloma initiation
dephosphorylation and inhibition of mitogen-activated
protein kinase in human mononuclear phagocytes. Role of
the Src homology 2 containing tyrosine phosphatase 1.
J Biol Chem 273: 645–652.
Kolk, A.H., Ho, M.L., Klatser, P.R., Eggelte, T.A., Kuijper, S.,
de Jonge, S., and van Leeuwen, J. (1984) Production and
characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. Clin Exp
Immunol 58: 511–521.
Ludwiczak, P., Brando, T., Monsarrat, B., and Puzo, G. (2001)
Structural characterization of Mycobacterium tuberculosis
lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. Anal Chem 73: 2323–
2330.
Ludwiczak, P., Gilleron, M., Bordat, Y., Martin, C., Gicquel, B.,
and Puzo, G. (2002) Mycobacterium tuberculosis phoP
mutant: lipoarabinomannan molecular structure. Microbiology 148: 3029–3037.
Lugo-Villarino, G., Balla, K.M., Stachura, D.L., Banuelos, K.,
Werneck, M.B., and Traver, D. (2010) Identification of
dendritic antigen-presenting cells in the zebrafish. Proc
Natl Acad Sci USA 107: 15850–15855.
Maeda, N., Nigou, J., Herrmann, J.L., Jackson, M., Amara,
A., Lagrange, P.H., et al. (2003) The cell surface receptor
DC-SIGN discriminates between Mycobacterium species
through selective recognition of the mannose caps on
lipoarabinomannan. J Biol Chem 278: 5513–5516.
Mishra, A.K., Driessen, N.N., Appelmelk, B.J., and
Besra, G.S. (2011a) Lipoarabinomannan and related
glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host-pathogen
interaction. FEMS Microbiol Rev 35: 1126–1157.
Mishra, A.K., Krumbach, K., Rittmann, D., Appelmelk, B.,
Pathak, V., Pathak, A.K., et al. (2011b) Lipoarabinomannan
biosynthesis in Corynebacterineae: the interplay of two
alpha(1–>2)-mannopyranosyltransferases MptC and MptD
in mannan branching. Mol Microbiol 80: 1241–1259.
Mishra, A.K., Alves, J.E., Krumbach, K., Nigou, J., Castro,
A.G., Geurtsen, J., et al. (2012) Differential arabinan
capping of lipoarabinomannan modulates innate immune
responses and impacts T helper cell differentiation. J Biol
Chem 287: 44173–44183.
Nigou, J., Gilleron, M., Cahuzac, B., Bounery, J.D., Herold,
M., Thurnher, M., and Puzo, G. (1997) The phosphatidylmyo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis bacillus Calmette Guerin. Heterogeneity,
structure, and role in the regulation of cytokine secretion.
J Biol Chem 272: 23094–23103.
Nigou, J., Gilleron, M., and Puzo, G. (1999)
Lipoarabinomannans: characterization of the multiacylated
forms of the phosphatidyl-myo-inositol anchor by NMR
spectroscopy. Biochem J 337 (Part 3): 453–460.
Nigou, J., Zelle-Rieser, C., Gilleron, M., Thurnher, M., and
Puzo, G. (2001) Mannosylated lipoarabinomannans inhibit
IL-12 production by human dendritic cells: evidence for a
negative signal delivered through the mannose receptor.
J Immunol 166: 7477–7485.
Nigou, J., Gilleron, M., and Puzo, G. (2003) Lipoarabinomannans: from structure to biosynthesis. Biochimie
85: 153–166.
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108
2107
Nigou, J., Vasselon, T., Ray, A., Constant, P., Gilleron, M.,
Besra, G.S., et al. (2008) Mannan chain length controls
lipoglycans signaling via and binding to TLR2. J Immunol
180: 6696–6702.
Pathak, S.K., Basu, S., Bhattacharyya, A., Pathak, S., Kundu,
M., and Basu, J. (2005) Mycobacterium tuberculosis
lipoarabinomannan-mediated IRAK-M induction negatively
regulates Toll-like receptor-dependent interleukin-12 p40
production in macrophages. J Biol Chem 280: 42794–
42800.
Pitarque, S., Herrmann, J.L., Duteyrat, J.L., Jackson, M.,
Stewart, G.R., Lecointe, F., et al. (2005) Deciphering the
molecular bases of Mycobacterium tuberculosis binding to
the lectin DC-SIGN reveals an underestimated complexity.
Biochem J 392: 615–624.
Prouty, M.G., Correa, N.E., Barker, L.P., Jagadeeswaran, P.,
and Klose, K.E. (2003) Zebrafish-Mycobacterium marinum
model for mycobacterial pathogenesis. FEMS Microbiol
Lett 225: 177–182.
Quesniaux, V.J., Nicolle, D.M., Torres, D., Kremer, L.,
Guerardel, Y., Nigou, J., et al. (2004) Toll-like receptor
2 (TLR2)-dependent-positive and TLR2-independentnegative regulation of proinflammatory cytokines by
mycobacterial lipomannans. J Immunol 172: 4425–
4434.
Rajaram, M.V., Ni, B., Morris, J.D., Brooks, M.N., Carlson,
T.K., Bakthavachalu, B., et al. (2011) Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating
macrophage MAPK-activated protein kinase 2 (MK2) and
microRNA miR-125b. Proc Natl Acad Sci USA 108: 17408–
17413.
Ramakrishnan, L., Valdivia, R.H., McKerrow, J.H., and
Falkow, S. (1997) Mycobacterium marinum causes both
long-term subclinical infection and acute disease in the
leopard frog (Rana pipiens). Infect Immun 65: 767–
773.
van der Sar, A.M., Musters, R.J., van Eeden, F.J., Appelmelk,
B.J., Vandenbroucke-Grauls, C.M., and Bitter, W. (2003)
Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections. Cell Microbiol 5:
601–611.
van der Sar, A.M., Abdallah, A.M., Sparrius, M., Reinders, E.,
Vandenbroucke-Grauls, C.M., and Bitter, W. (2004) Mycobacterium marinum strains can be divided into two distinct
types based on genetic diversity and virulence. Infect
Immun 72: 6306–6312.
Sena, C.B., Fukuda, T., Miyanagi, K., Matsumoto, S.,
Kobayashi, K., Murakami, Y., et al. (2010) Controlled
expression of branch-forming mannosyltransferase is critical for mycobacterial lipoarabinomannan biosynthesis.
J Biol Chem 285: 13326–13336.
Sibley, L.D., Hunter, S.W., Brennan, P.J., and Krahenbuhl,
J.L. (1988) Mycobacterial lipoarabinomannan inhibits
gamma interferon-mediated activation of macrophages.
Infect Immun 56: 1232–1236.
Stamm, L.M., Morisaki, J.H., Gao, L.Y., Jeng, R.L.,
McDonald, K.L., Roth, R., et al. (2003) Mycobacterium
marinum escapes from phagosomes and is propelled by
actin-based motility. J Exp Med 198: 1361–1368.
Stinear, T.P., Seemann, T., Harrison, P.F., Jenkin, G.A.,
Davies, J.K., Johnson, P.D., et al. (2008) Insights from the
2108 E. J. M. Stoop et al.
complete genome sequence of Mycobacterium marinum
on the evolution of Mycobacterium tuberculosis. Genome
Res 18: 729–741.
Stoop, E.J., Schipper, T., Huber, S.K., Nezhinsky, A.E.,
Verbeek, F.J., Gurcha, S.S., et al. (2011) Zebrafish
embryo screen for mycobacterial genes involved in
the initiation of granuloma formation reveals a newly
identified ESX-1 component. Dis Model Mech 4: 526–
536.
Swaim, L.E., Connolly, L.E., Volkman, H.E., Humbert, O.,
Born, D.E., and Ramakrishnan, L. (2006) Mycobacterium
marinum infection of adult zebrafish causes caseating
granulomatous tuberculosis and is moderated by adaptive
immunity. Infect Immun 74: 6108–6117.
Tailleux, L., Schwartz, O., Herrmann, J.L., Pivert, E.,
Jackson, M., Amara, A., et al. (2003) DC-SIGN is the major
Mycobacterium tuberculosis receptor on human dendritic
cells. J Exp Med 197: 121–127.
Talaat, A.M., Reimschuessel, R., Wasserman, S.S., and
Trucksis, M. (1998) Goldfish, Carassius auratus, a novel
animal model for the study of Mycobacterium marinum
pathogenesis. Infect Immun 66: 2938–2942.
Tatituri, R.V., Illarionov, P.A., Dover, L.G., Nigou, J., Gilleron,
M., Hitchen, P., et al. (2007) Inactivation of Corynebacterium glutamicum NCgl0452 and the role of MgtA
in the biosynthesis of a novel mannosylated glycolipid
involved in lipomannan biosynthesis. J Biol Chem 282:
4561–4572.
Torrelles, J.B., Azad, A.K., and Schlesinger, L.S. (2006) Fine
discrimination in the recognition of individual species of
phosphatidyl-myo-inositol mannosides from Mycobacterium tuberculosis by C-type lectin pattern recognition
receptors. J Immunol 177: 1805–1816.
Torrelles, J.B., Sieling, P.A., Arcos, J., Knaup, R., Bartling, C.,
Rajaram, M.V., et al. (2011) Structural differences in
lipomannans from pathogenic and nonpathogenic mycobacteria that impact CD1b-restricted T cell responses.
J Biol Chem 286: 35438–35446.
Vergne, I., Chua, J., and Deretic, V. (2003) Tuberculosis toxin
blocking phagosome maturation inhibits a novel Ca2+/
calmodulin-PI3K hVPS34 cascade. J Exp Med 198: 653–
659.
Vignal, C., Guerardel, Y., Kremer, L., Masson, M., Legrand,
D., Mazurier, J., and Elass, E. (2003) Lipomannans, but
not lipoarabinomannans, purified from Mycobacterium
chelonae and Mycobacterium kansasii induce TNFalpha and IL-8 secretion by a CD14-toll-like receptor
2-dependent mechanism. J Immunol 171: 2014–
2023.
Volkman, H.E., Clay, H., Beery, D., Chang, J.C., Sherman,
D.R., and Ramakrishnan, L. (2004) Tuberculous granuloma formation is enhanced by a mycobacterium virulence
determinant. PLoS Biol 2: e367.
van der Wel, N., Hava, D., Houben, D., Fluitsma, D., van Zon,
M., Pierson, J., et al. (2007) M. tuberculosis and M. leprae
translocate from the phagolysosome to the cytosol in
myeloid cells. Cell 129: 1287–1298.
Wittamer, V., Bertrand, J.Y., Gutschow, P.W., and Traver, D.
(2011) Characterization of the mononuclear phagocyte
system in zebrafish. Blood 117: 7126–7135.
Wu, T., Guo, S., Wang, J., Li, L., Xu, L., Liu, P., et al. (2011)
Interaction between mannosylated lipoarabinomannan and
dendritic cell-specific intercellular adhesion molecule-3
grabbing nonintegrin influences dendritic cells maturation
and T cell immunity. Cell Immunol 272: 94–101.
Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Table S1. Antibiotic susceptibility of mptC mutant.
Fig. S1. Schematic structure of lipoarabinomannan (LAM) of the
different strains used in this study. Schematic representations of
LAM of wildtype M. marinum, the mptC mutant, the capless
mutant, the mptC mutant complemented with the M. marinum or
M. tuberculosis mptC (mptC mutant + Mm/Mtb-mptC), or the
mptC mutant complemented with the M. smegmatis mptC
(mptC mutant + Msm-mptC). ManLAM is anchored into the
mycobacterial cell envelope by its mannosylphosphatidyl-myoinositol (MPI) moiety. The MPI anchor is linked to a mannan core
consisting of α(1→6)-linked mannopyranose (manp) residues
branched with α(1→2)-linked manp units. The structure is further
glycosylated with an arabinan domain consisting of a linear
α(1→5)-linked arabinofuranosyl (araf) polymer branched with
linear or bifurcating araf side-chains. The non-reducing termini of
the arabinan domain can be substituted with one to three manp
residues; the mannose caps. The first manp unit is α(1→5)-linked
and the following manp residues are α(1→2)-linked.
Fig. S2. α(1→2)-Mannosyltransferase activity of mptC mutant
and related strains. In vitro α(1→2)-mannosyltransferase assay
with synthetic nonasaccharide acceptor and membrane fractions
from the blank (control), wildtype M. marinum (WT), mptC
mutant (mptC), mptC mutant complemented with mmar_3225
(mptC + mptC Mm), mptC mutant complemented with
msmeg4247 (mptC + mptC Msm) and mptC mutant complemented with Rv2181 (mptC + mptC Mtb). The results represent
mean + SEM of three independent experiments.
Fig. S3. Analysis of PIM biosynthesis in wildtype M. marinum
(wildtype) and the mptC mutant (mptC). The polar lipids were
extracted and examined by 2D-TLC. First and second dimensions are indicated by arrows and numbers.
Fig. S4. EM images of sections of THP-1 cells infected for 24 h
with the M. marinum mptC mutant. Representative images of
sections of cells with (A) cytosolic and (B) phagosomal mptC
mutant bacterium (Mm), and immunogold labelled for CD63 with
10 nm gold particles, indicating the presence of phagosomal
membranes. L, lysosome; M, mitochondria; N, nucleus; and the
bars represent 200 nm.
© 2013 John Wiley & Sons Ltd, Cellular Microbiology, 15, 2093–2108